Insulin antagonism of glucocorticoid induction of tyrosine aminotransferase in cultured foetal hepatocytes

Polyadenylated RNA from developing Artemia salina cysts was fractionated by centrifugation through a sucrose gradient containing methylmercuric hydroxide (CH3HgOH). Aliquots of each fraction were directly added to a rabbit reticulocyte lysate to program protein synthesis in vitro. The translation products were assayed for eukaryotic elongation factor Tu (eEF-Tu) by immunoprecipitation with an antibody raised in rabbits and purified by affinity chromatography. The immunoprecipitated radioactivity was analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate. Sequences coding for eEF-Tu sediment in the 20-S region of the gradient and form a major component of the poly(A)-containing RNA. The mRNA of the 20-S region, comprising about 10% of the poly(a)-containing RNA fractionated on the gradient, has been translated in vitro and 30% of the translation products represent immunoprecipitable eEF-Tu protein chains with an Mr of 50000.     

Messenger RNA during early embryogenesis in Artemia salina: altered translatability and sequence complexity

RNA from developing embryos of Artemia salina (5, 10, and 20 h after re-initiation of development) was translated 3-10 times more efficiently in a rabbit reticulocyte lysate cell-free protein synthesizing system than RNA from dormant gastrulae. The latter did not appear to contain any significant amount of translation inhibitor activity. Ninety percent of the translatable activity in dormant gastrulae was recovered as poly(A)--RNA, whereas 80% of that in post-gastrular developing embryos was present as poly(A)+-RNA. The size of most polypeptides coded for by dormant gastrular RNA was less than 130,000 daltons whereas the size of those coded for by developing embryonic RNA was up to 200,000 daltons, which correlated with a corresponding shift to poly A-containing RNA of higher molecular weight. Two major polypeptides of about 37,000 daltons coded for by dormant gastrular RNA disappeared at 20 h after resumption of development. Hybridization of complementary DNA (cDNA) to a 1000-fold excess of the homologous poly(A)+-RNA revealed the presence of three complexity classes of mRNA. Forty-five percent, 30%, and 25% of RNA in dormant gastrulae were present as high, middle, and low abundance classes comprising about 10, 80, and 9700 species, respectively whereas in the nauplii there were 10, 150, and 7900 species of high, middle, and low abundancy sequences, respectively. Heterologous hybridizations using cDNA complementary to highly abundant messenger population of nauplii (isolated by chromatography on hydroxyapatite) to poly(A)+-RNA from dormant cysts showed considerably divergence in this class of messengers from the two developmental stages. Re-initiation of development of dormant Artemia gastrulae is thus characterized by a "re-programming" seen as a simultaneous and rapid increase in the polyadenylation and translatability of poly(A)+-RNA accompanied by a qualitative change in its sequence complexity.     

Cloning of cDNA sequences for an Artemia salina hnRNP protein: evidence for conservation through evolution.

A cDNA clone was isolated for Artemia salina protein HD40, a component of heterogenous nuclear ribonucleoproteins. Enriched Artemia 15S poly(A)+ RNA was used as a template and double-stranded cDNA sequences were inserted into the Pst I restriction endonuclease site of E. coli plasmid pBR322. Recombinant colonies were analyzed by positive hybrid selection of poly(A)+ RNA that directs the synthesis of protein HD40 in an in vitro assay. In vitro translation of the mRNA selected by recombinant clone 87HD yields a protein that is immunoprecipitated by anti-HD40 antibodies and that comigrates with authentic HD40 on gel electrophoresis. Partial proteolysis of protein HD40 and the in vitro translated product selected by clone 87HD produces the same peptide patterns. The size of the cloned insert is about 820 bp. The length of HD40 mRNA as determined by Northern blot analysis, is about 1500 nucleotides. Southern blot analysis performed with DNA of different species (plant, avian, mammal) shows cross-hybridizing bands when probed with clone 87HD DNA suggesting that the HD40 gene is evolutionarily conserved.     

Biochemical changes in progressive muscular dystrophy. XIV. Skeletal muscle myosin mRNA translatability in dystrophic mice

Variations in the content and translatability of the poly(A)+ RNA and mRNA molecules coding for myosin (M) were studied in the hind leg muscles of genetically dystrophic mice. The poly(A)+ RNA content of total skeletal muscle failed to increase normally during progression of the disease. M mRNA, isolated from dystrophic normally during progression of the disease. M mRNA, isolated from dystrophic murine muscle poly(A)+ RNA, was mostly found to be associated with the 26S RNA species. The translation of M mRNA in an in vitro heterologous wheat germ system was lower at 8 and 16 weeks in the dystrophic group as compared with the controls. Analysis of the translation products via sodium dodecyl sulfate-polyacrylamide gel electrophoresis, autoradiography, and densitometric autoradiographic tracing demonstrated the gradual disappearance of a protein band corresponding to M, the major component of skeletal muscle. cDNA was synthesized, using M mRNA that was isolated and purified from normal and dystrophic mouse muscle as a template. Total radioactivity was measured in some cDNA fractions produced from normal and dystrophic mouse muscle, while other fractions were utilized for separation and sizing of cDNA by disc gel electrophoresis. The cDNA from normal muscle was hybridized with M mRNA from normal and 16-week-old dystrophic mouse muscles. The cDNA probe, hybridization experiments, and studies involving the content and synthesis of M mRNA suggest that murine muscular dystrophy elicited a shorter species of mRNA or shorter sequences of the same species of mRNA coding for M. Not all poly(A)+ mRNA sequences coding for M, found in control mice, were present in their dystrophic counterparts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of the membrane-associated poly(A)+RNA in the cytoplasm of dormant Artemia cysts by DNA excess hybridization. Evidence for a nuclear origin

Encysted embryos of Artemia contain latent mRNA, to a large extent associated with a fraction of cytoplasmic membranes. The membranes, purified by EDTA treatment and banding in a sucrose gradient at 1.17 g/cm3, include endoplasmic vesicles and mitochondria. The origin of the membrane-associated poly(A)+RNA was therefore investigated. In gel electrophoresis poly(A)+RNA from the purified membranes of dormant cysts forms two distinct bands at approx. 3 . 10(5) and 5 . 10(5) Da. Later during development the lighter component decreases. Nuclei from dormant cysts are devoid of poly(A)+RNA, while nuclei from developing embryos (50% emergence) contain a predominant poly(A)+RNA component of approx. 5 . 10(5) Da. 125I-labelled preparations of nuclear DNA and of nuclear and membrane-associated poly(A)+RNA were used in reassociation and hybridization experiments with excess nuclear DNA. Poly(A)+RNA from the membranes of dormant cysts hybridized to nuclear DNA to the same extent as the nuclear poly(A)+RNA from developing embryos. The hybridization of labelled, nuclear poly(A)+RNA to nuclear DNA was strongly inhibited by unlabelled membrane RNA from either dormant cysts or developing embryos. It is concluded that the stored, membrane-associated poly(A)+RNA in dormant cysts is essentially of nuclear origin. The 5 . 10(5)-Da component is largely homologous with the corresponding component of nuclear poly(A)+RNA at later stages.     

Binding of eucaryotic elongation factor Tu to nucleic acids

The binding of eucaryotic elongation factor Tu (eEF-Tu) to nucleic acids was investigated. eEF-Tu binds to a variety of different nucleic acids with high affinity, showing a strong preference for 18 S and 28 S rRNA over transfer RNA and for ribose-containing polymers over polydeoxyribonucleotides. The factor binds at multiple sites on 28 S rRNA without strong cooperativity. eEF-Tu binds strongly to poly(G) and poly(U) but weakly, if at all, to poly(A) and poly(C). Experiments employing an airfuge demonstrate that eEF-Tu can form a quaternary complex containing the factor, 28 S rRNA, aminoacyl-tRNA, and GTP. The existence of two distinct RNA binding sites on eEF-Tu suggests that rRNA may play a role in the recognition of eEF-Tu.aminoacyl-tRNA.GTP complexes by polysomes. Support for this suggestion comes from experiments which show that poly(G) inhibits the factor-dependent binding of aminoacyl-tRNA to mRNA-programmed 80 S ribosomes. In addition, it is shown that eEF-Tu possesses an intrinsic GTPase activity which is stimulated significantly by 28 S rRNA, poly(G), and poly(U). The binding of eEF-Tu to poly(G) lowers the activation energy for eEF-Tu GTPase from 74.3 to 65.9 kJ . mol-1 and approximately doubles the Vmax of the enzymatic reaction. The results are discussed in relation to the binding of eEF-Tu to ribosomes during protein synthesis.     

Structure and function of rat liver polysome populations. II. Characterization of polyadenylate-containing mRNA associated with subpopulations of membrane-bound particles

Poly(A)+RNA fractions prepared from free and loosely and tightly membrane-bound polysome populations (poly(A)+RNAfree, poly(A)+RNAloose, and poly(A)+RNAtight) were used to drive cDNA in homologous and heterologous hybridization reactions. A large fraction by mass of sequences was shared among the three poly(A)+RNA populations, but shared sequences exhibited distinct frequency distributions within the different populations. 13-15 in vitro translation products of poly(A)+RNAfree and poly(A)+RNAloose detected by gel electrophoresis were shared. Most of these were produced in different relative quantities by the two RNA populations. Five or six higher mol wt polypeptides were produced by poly(A)+RNAloose that were not detected as products of either poly(A)+free or poly(A)+RNAtight. We suggest that loosely bound polysomes may not be artifactually derived as reflected in their quantitatively distinct poly(A)+RNA population. Two tightly membrane-bound RNP fractions were prepared from rat liver on the basis of their release from or retention on purified rough microsomes or a crude membrane fraction after in vitro disaggregation of polysomes with high-salt and puromycin. Homologous and heterologous hybridizations involving their poly(A)+RNA fractions revealed that a large portion by mass of sequences was shared but that these sequences exhibited distinct frequency distributions in the two fractions. The RNA fractions produced exhibited distinct frequency distributions in the two fractions. The RNA fractions produced an identical set of in vitro translation products but individual polypeptides were produced in different relative quantities. This indicates that the two RNP fractions do not arise by any random artifactual process and suggests that they may represent functionally distinct populations.     

Affinity labeling of eukaryotic elongation factors using N epsilon-bromoacetyl-Lys-tRNA.

eEF-T and eEF-Tu from rabbit reticulocyte and from Artemia were affinity labeled using N epsilon-bromoacetyl-Lys-tRNA prepared with either yeast or E. coli tRNA. Only the eEF-Tu polypeptide was crosslinked when eEF-T was incubated with the reactive aminoacyl-tRNA analogue, which indicates that at least part of the aminoacyl-tRNA binding site is the same in both eEF-Tu and the multisubunit eEF-T. Complex formation (eEF-Tu x aa-tRNA x GTP) was required for crosslinking, since no covalent reaction with eEF-Tu occurred in the absence of GTP. The yield of crosslinked product was greatly reduced by adding either unmodified rabbit liver aminoacyl-tRNA or unmodified E. coli Lys-tRNA to the incubation to compete for the aminoacyl-tRNA binding site on eEF-T or eEF-Tu, indicating that the covalent reaction occurs while the N epsilon-bromoacetyl-Lys-tRNA is bound in this site. The affinity labeling of a prokaryotic and two different eukaryotic elongation factors by the same reagent suggests that there may be conservation of structure in the region of the proteins which binds the aminoacyl end of the aminoacyl-tRNA.     

The stability of mRNA for eucaryotic elongation factor Tu in Friend erythroleukemia cells varies with growth rate.

The decay rates of eucaryotic elongation factor Tu (eEF-Tu) mRNA and eucaryotic initiation factor 4A (eIF-4A) mRNA in Friend erythroleukemia (FEL) cells were determined under several different growth conditions. In FEL cells which were no longer actively dividing (stationary phase), eEF-Tu mRNA was found to be rather stable, with a t1/2 of about 24 h. In rapidly growing FEL cells eEF-Tu mRNA was considerably less stable, with a t1/2 of about 9 h. In both cases a single rate of mRNA decay was observed. However, when stationary-phase cells resumed growth after treatment with fresh medium, we observed that eEF-Tu mRNA decay followed a biphasic process. The faster of the two decay rates involved approximately 50% of the eEF-Tu mRNA and had a t1/2 of about 1 h. The decay rates for eIF-4A (t1/2 = 2 h) and total poly(A)+ RNA (t1/2 = 3 h) were unaffected by changes in growth conditions. The t1/2 for polysomal eEF-Tu mRNA was found to be about 8 h when stationary FEL cells were treated with fresh medium. Previous work in this laboratory has shown (T. R. Rao and L. I. Slobin, Mol. Cell. Biol. 7:687-697, 1987) that when FEL cells are allowed to grow to stationary phase, approximately 60% of the mRNA for eEF-Tu is found in a nontranslating postpolysomal messenger ribonucleoprotein (mRNP) particle. eEF-Tu mRNP was rapidly cleared from stationary cells after treatment with fresh medium. The data presented in this report indicate that the stability of eEF-Tu mRNP is rapidly altered and the particle is targeted for degradation when stationary FEL cells resume growth.     

Thyroxine increases neonatal mouse submandibular gland mRNA-directed synthesis of epidermal growth factor

Thyroid hormones are known to modulate the concentrations of epidermal growth factor (EGF) in the mouse submandibular gland (SMG); this action is presumably mediated by the nuclear triiodothyronine receptor. To test the hypothesis that thyroid hormones act to increase SMG EGF concentrations by increasing the number of poly(A)+ -specific mRNA, poly(A)+ RNA was isolated from SMGs of neonatal mice which had been treated daily from birth through to 21 days of age with thyroxine (T4,0.4 microgram/g body weight). Poly(A)+ RNA also was extracted from SMGs of intact 21-day-old mice which had received vehicle alone. No significant differences in total nucleic acid, total RNA, or poly(A)+ RNA yields were noted between the two groups of animals. The isolated poly(A)+ RNAs from T4-treated and control mice were translated in an in vitro wheat germ system. Although no significant differences in efficiency of [35S]cysteine incorporation into trichloracetic acid precipitable material were noted between the two poly(A)+ RNA preparations, a significantly greater proportion of radioactivity was immunoprecipitable by anti-EGF antiserum in the translation medium derived from T4-treated mice (17.2 +/- 0.9%, mean +/- SEM) than in that of control mice (7.3 +/- 0.5%, P less than 0.001). Polyacrylamide gel electrophoresis of the immunoprecipitates (IMMP) revealed the presence of three radioactive bands with apparent relative masses (MrS) of 12,000, 9000, and 6000. The latter species comigrated with purified EGF, [125I]EGF, and an IMMP of a SMG extract. The translation product IMMPs following polyacrylamide gel electrophoresis were iodinated and digested with alpha-chymotrypsin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning and sequencing of the cDNA for S-acyl fatty acid synthase thioesterase from the uropygial gland of mallard duck

In vitro translation of poly(A)+ RNA from the uropygial glands of mallard ducks (Anas platyrhynchos) generated a 29-kDa protein which cross-reacted with rabbit antibodies prepared against S-acyl fatty acid synthase thioesterase (Kolattukudy, P. E., Rogers, L., and Flurkey, W. (1985) J. Biol. Chem., 260, 10789-10793). A poly(A)+ RNA fraction enriched in this thioesterase mRNA, isolated by sucrose density gradient centrifugation, was used to prepare cDNA which was cloned in Escherichia coli using the plasmid pUC9. Using hybrid-selected translation and colony hybridization, 17 clones were selected which contained the cDNA for S-acyl fatty acid synthase thioesterase. Northern blot analysis showed that the mature mRNA for this thioesterase contained 1350 nucleotides whereas the cloned cDNA inserts contained 1150-1200 base pairs. Five of the 6 clones tested for 5'-sequence had identical sequences, and the three tested for 3'-end showed the same sequence with poly(A) tails. Two clones, pTE1 and pTE3, representing nearly the full length of mRNA, were selected for sequencing. Maxam-Gilbert and Sanger dideoxy chain termination methods were used on the cloned cDNA and on restriction fragments subcloned in M13 in order to determine the complete nucleotide sequence of the cloned cDNA. The nucleotide sequence showed an open reading frame coding for a peptide of 28.8 kDa. Two peptides isolated from the tryptic digest of the thioesterase purified from the gland showed amino acid sequences which matched with two segments of the sequence deduced from the nucleotide sequence. Another segment containing a serine residue showed an amino acid sequence homologous to the active serine-containing segment of the thioesterase domain of fatty acid synthase. Thus, the clones represent cDNA for S-acyl fatty acid synthase thioesterase. The present results constitute the first case of a complete sequence of a thioesterase.     

Polyadenylated mRNA from the photosynthetic procaryote Rhodospirillum rubrum.

Total cellular RNA extracted from Rhodospirillum rubrum cultured in butyrate-containing medium under strict photosynthetic conditions to the stationary phase of growth has been fractionated on an oligodeoxythymidylic acid-cellulose column into polyadenylated [poly(A)+] RNA and poly(A)- RNA fractions. The poly(A)+ fraction was 9 to 10% of the total bulk RNA isolated. Analysis of the poly(A)+ RNA on a denaturing urea-polyacrylamide gel revealed four sharp bands of RNA distributed in heterodisperse fashion between 16S and 9S. Similar fractionation of the poly(A)- RNA resulted in the separation of 23, 16, and 5S rRNAs and 4S tRNA. Poly(A)+ fragments isolated after combined digestion with pancreatic A and T1 RNases and analysis by denaturing gel electrophoresis demonstrated two major components of 80 and 100 residues. Alkaline hydrolysis of the nuclease-resistant, purified residues showed AMP-rich nucleotides. Through the use of snake venom phosphodiesterase, poly(A) tracts were placed at the 3' end of poly(A)+ RNA. Stimulation of [3H]leucine incorporation into hot trichloroacetic acid-precipitable polypeptides in a cell-free system from wheat germ primed by the poly(A)+ RNA mixture was found to be 220-fold higher than that for poly(A)- RNAs (on a unit mass basis), a finding which demonstrated that poly(A)+ RNAs in R. rubrum are mRNAs. Gel electrophoretic analysis of the translation mixture revealed numerous 3H-labeled products including a major band (Mr, 52,000). The parent protein was precipitated by antibodies to ribulose bisphosphate carboxylase-oxygenase and comprised 6.5% of the total translation products.     

Molecular induction by phenobarbital of a rat hepatic form of cytochrome P-450: expression of a 4-kilobase messenger RNA

A differential screening procedure was employed to isolate a cDNA clone corresponding to a major phenobarbital (PB)-inducible form of rat hepatic cytochrome P-450. The G-C homopolymer-tailing technique was utilized to construct a cDNA library in the PstI site of plasmid pBR322. The library represented PB-induced poly(A+)RNA sequences from hepatic polysomes of 150-g male Sprague-Dawley rats. Hybrid-selection experiments against total PB-inducible RNA were performed with plasmid DNA derived from clones enriched in PB-inducible information. The mRNA molecules that specifically hybridized were subjected to in vitro translation, were immunoprecipitated with antibody raised in rabbits against purified cytochrome P-450b (P. E. Thomas, D. Korzeniowski, D. Ryan, and W. Levin (1979) Arch. Biochem. Biophys. 192, 524-532), and were electrophoresed under sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoretic conditions. One cDNA clone, designated PB-8, contained a 600-bp insert partially coding for a PB-inducible cytochrome P-450 species that comigrated on SDS-gel electrophoresis with highly purified P-450b. A single injection of PB, 15-18 h before sacrifice, increased the level of polysomal poly(A+)RNA complementary to the isolated cDNA clone by approximately 16-fold. Northern blot hybridizations of polysome-derived poly (A+)RNA, electrophoresed in denaturing agarose gels, demonstrated that the size of the mRNA corresponding to the isolated clone was 4 kb. Isolated heteronuclear RNA species demonstrated a time-dependent increase in the synthesis of a similar 4-kb RNA molecule. By genomic blot hybridization to EcoRI-restricted DNA, at least three complementary DNA fragments migrating at 5.1, 3.2, and 2.9 kb were observed with 32P-labeled PB-8 as a probe. These data, together with restriction endonuclease mapping and partial cDNA sequence information of the PB-8 cDNA, suggest that the PB-8 clone represents a previously unreported cDNA clone for a form of cytochrome P-450 inducible by PB.     

Molecular cloning and sequencing of cDNA encoding goat pre alpha-lactalbumin

In poly(A)+RNA extracted from a lactating goat mammary gland, mRNA of about 750 nucleotides was shown to encode pre alpha-lactalbumin by using in vitro translation and immunoprecipitation. From the total poly(A)+RNA, the cDNA library was constructed using the Escherichia coli plasmid pUC18; it was screened with the oligodeoxyribonucleotide probe corresponding to the amino acid sequence of Trp60-Gln65 of goat alpha-lactalbumin. A plasmid containing almost full-length cDNA of goat pre alpha-lactalbumin, pGLA-1, was identified. The cDNA insert of pGLA-1 comprises 727 base pairs and contains the signal peptide and mature protein sequence.     

Biochemical studies of mammalian oogenesis: possible existence of a ribosomal and poly(A)-containing RNA-protein supramolecular complex in mouse oocytes

Mouse follicles were labeled with [3H]uridine and then cultured in vitro for 3 days. When oocytes were disrupted, about 40% of the total radiolabeled RNA could be sedimented at 9,000g. Fractionation of this RNA on poly(U)-Sepharose revealed that about 30% and 60% of the total amount of radiolabeled poly(A)- and poly(A)+ RNA, respectively, were in the pellet fraction. Treatments that disrupt protein structure reduced the amount of 9,000g sedimentable RNA and affected to the same extent the distribution of Poly(A)- and poly(A)+ RNA in the pellet and supernatant fractions. CsCl centrifugation of formaldehyde-fixed pellets revealed that virtually all of the radiolabeled RNA had a density significantly lower than that of ribosomes. The sedimentable RNA appeared not to be polysomal, membrane bound or associated wih a cytoskeleton. Agarose gel electrophoresis after poly(U)-Sepharose fractionation of either the pellet or supernatant revealed the presence of 28S, 18S, 5S + 4S, and heterodisperse poly(A)+ RNA. The size of distribution of poly(A)+ RNA in the pellet and supernatant fractions was fairly similar. Pulse-chase experiments revealed that the stability of poly(A)- RNA in the pellet and supernatant fractions was the same within the experimental error and a similar situation was found for poly(A)+ RNA. RNA in pellet translated in vitro coded for discrete size classes of protein. Since the relative band intensities were similar for both total and pellet RNA translated in vitro there seemed to be no major partitioning of specific size classes of mRNA into the pellet fraction. These results are discussed in terms of a possible composition of the lattice structures that accumulate during mouse oocyte growth and have been postulated to be a storage form for ribosome (Burkholder et al., '71).     

Patterns of maternal messenger RNA accumulation and adenylation during oogenesis in Urechis caupo

We have investigated the accumulation and adenylation of the maternal mRNA during oogenesis in the oocytes of the marine worm Urechis caupo. The analysis, using in vitro translation and cDNA probes to assay for specific mRNAs, demonstrates that different maternal mRNAs accumulate with different patterns during oogenesis. One class of maternal mRNAs accumulates throughout oogenesis and remains at a steady level in the full-grown oocyte. These mRNAs do not have a poly(A) tail long enough to mediate binding to oligo(dT)-cellulose in oocytes, but are rapidly adenylated immediately following fertilization. The other maternal mRNAs accumulate in growing oocytes as poly(A)+ RNA and undergo some deadenylation in full-grown oocytes and embryos. Some of these mRNAs attain their highest concentration fairly early in oogenesis, while others continue to accumulate during later stages. Many of the mRNAs that accumulate as poly(A)+ RNA in growing oocytes diminish dramatically in concentration in full-grown oocytes.     

Isolation of a cloned DNA segment containing a ribosomal protein gene of Drosophila melanogaster

A Drosophila genomic DNA library in the vector Charon 4 was screened using cDNA derived from the small (6S-12S) poly(A)+ mRNA of 2-6-h-old Drosophila embryos. This fraction of mRNA is enriched for ribosomal protein-coding sequences. The selected recombinants were hybridized to total mRNA under conditions which allowed for isolation of homologous mRNAs. The mRNA from these RNA/DNA hybrids was eluted and translated in vitro. The translation products were analyzed by one- and two-dimensional electrophoresis with authentic ribosomal proteins as standards. One cloned DNA segment was found to contain a ribosomal protein gene, and a sequence which hybridizes strongly to at least 5 other ribosomal protein mRNAs.     

Eucaryotic elongation factors Ts is an integral component of rabbit reticulocyte elongation factor 1.

Elongation factor 1 (EF-1) was purified from rabbit reticulocytes and found to contain at least two distinct polypeptides: one of Mr 53 000 and one of Mr 30 000. The 30 000-Mr polypeptide was purified from EF-1 by treatment of the factor with 5.4 M guanidine . HCl and subsequent chromatography on DEAE-BioGel A in the presence of 5 M urea. By a number of functional criteria, the 30 000-Mr polypeptide was found to be the eucaryotic elongation factor Ts (eEF-Ts). These criteria include the ability of the polypeptide to stimulate Artemia salina eEF-Tu-dependent binding of aminoacyl-tRNA to 80-S ribosomes as well as eEF-Tu + EF-2-dependent polyphenylalanine synthesis. The reticulocyte factor also markedly increased the rate of exchange of eEF-Tu . gdp complexes with free GTP. Furthermore, rabbit antibodies to EF-1 from A. salina which was previously shown to contain eEF-Ts [Slobin, L. I. and M?ller, W. (1978) Eur. J. Biochem. 84, 69--77] were found to cross-react with reticulocyte eEF-Ts, suggesting extensive structural homology between brine shrimp and rabbit eEF-Ts. The demonstration that eEF-Ts is and integral component of EF-1 from such diverse sources as brine shrimp and rabbit reticulocytes supports the conclusion that the factor is universally present in eucaryotic EF-1.